Maxim, a traditional Chinese herbal medicine, has been widely used for tonifying kidneys and strengthening bone for thousands of years in China, Korea and Japan (5–7). Icariin (C₃₃H₄₀O₁₅; molecular weight, 676.67; Fig. 1), a flavonoid isolated from Epimedium brevicornum Maxim, is considered as the main pharmacological active constituent (8,9) and has been reported to possess various pharmacological effects, including anti-inflammatory, anti-osteoporosis (10), anti-tumor (11), immunoregulatory (12) and anti-oxidative actions (13). In addition, icariin has been shown to have beneficial effects on cardiovascular diseases such as atherosclerosis (14,15). However, the potential mechanisms of action of icariin against atherosclerosis have remained to be fully elucidated. In view of this, the present study was designed to elucidate whether icariin can attenuate the initiation and progression of atherosclerosis. The effects of icariin on ox-LDL-induced proliferation of VSMCs were assessed, and the results indicated that they are mediated via suppression of cell-cycle regulatory protein proliferating cell nuclear antigen (PCNA) and deactivation of ERK1/2.

Human aortic vascular smooth muscle cells (HA-VSMCs) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in 100-mm dishes in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sijiqing Bioengineering Material Co., Ltd. Hangzhou, China) and 1% penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

VSMCs in the logarithmic growth phase were seeded into 96-well plates at a density of 1×10⁴ cells per well and incubated for 24 h at 37°C in an atmosphere containing 5% CO₂. After pre-treatment with the indicated concentrations of icariin (purity, >98%; Vic's Biological Technology Co., Ltd, Sichuan, China; 0, 10, 20 or 40 µm) for 24 h prior to stimulation with oxidized low-density lipoprotein (ox-LDL; Yiyuan Biotechnologies Co., Ltd, Guangzhou, China; 50 µg/ml) for the indicated times (24 or 48 h). Subsequently, the medium was discarded and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT; Sigma-Aldrich) solution was added at a final concentration of 0.5 mg/ml, followed by incubation for 4 h at 37°C. The MTT solution was carefully removed and 150 µl dimethyl sulfoxide (Sigma-Aldrich) was added to each well followed by a 15-min incubation. The absorbance of each well was measured using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Inc.) with a reference wave length of 490 nm. The initial absorbance at 0 h, prior to icariin treatment, was also measured.

VSMCs in the logarithmic growth phase were seeded into six-well plates at a density of 1×10⁴ cells per well and then incubated for 24 h at 37°C in an atmosphere containing 5% CO₂. After pre-treatment with the indicated concentrations of icariin (0, 10, 20 or 40 µm) for 24 h, cells were incubated with or without ox-LDL (50 µg/ml) for a further 24 h. The cells were trypsinized, collected and washed twice with ice-cold phosphate-buffered saline (PBS) prior to fixing in 70% cold ethanol at 4°C overnight. Next, the fixed cells were re-suspended in PBS containing 100 µg/ml RNase A (Sigma-Aldrich) and 50 µg/ml propidium iodide (PI; Sigma-Aldrich) for 30 min at room temperature. Cells were then analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The percentages of cells in G0/G1, S and G2/M phases were determined using ModFit LT V3.3.11 software (Verity Software House Inc., Topsham, ME, USA).

VSMCs in the logarithmic growth phase were seeded into six-well plates for incubation for 24 h at 37°C in an atmosphere containing 5% CO₂. After pre-treatment with the indicated concentrations of icariin (0, 10, 20 or 40 µm) for 24 h, the cells were incubated with or without ox-LDL (50 µg/ml) for another 24 h. Subsequently, VSMCs were scraped in ice-cold PBS and lysed in cold lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). After centrifugation at 13,000 × g, the supernatant (total protein extract) was separated and quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein samples (50 µg) were loaded onto 10% SDS-PAGE gels (Applygen Technologies, Inc., Beijing, China) and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.05% Tween 20 (TBST; Zhongtian Jingwei Technologies, Inc., Beijing, China) and incubated with the primary rabbit anti-human monoclonal antibodies against ERK1/2 (dilution, 1:1,000; Abcam, Cambridge, MA, USA; cat. no. ab36911), anti-phosphorylated-ERK1/2 (dilution, 1:1,000; Abcam; cat. no. 50011) or anti-PCNA (dilution, 1:1,000; Abcam; cat. no. ab92552) or anti-GAPDH (dilution, 1:1,000; Sigma-Aldrich; cat. no. sab4300645) at 4°C overnight. Following incubation, the membranes were washed three times in TBST for 15 min and the membranes were incubated with horseradish-peroxidase-labeled goat anti-rabbit secondary antibody for 1 h at room temperature (dilution, 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-45101). Following three further washes in TBST, the protein expression levels were visualized using the enhanced chemiluminescence kit, BeyoECL Plus (Beyotime Institute of Biotechnology), images of the blots were captured on X-ray films (GE Healthcare, Little Chalfont, UK) and were analyzed using ImageJ version 1.46 (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as the protein loading control.

Statistical analysis was performed using the SPSS 17.0 statistical package (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. One-way analysis of variance was applied for multiple comparisons and the least significant difference test was applied for intra-group comparison. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of icariin on the proliferation of VSMCs induced by ox-LDL, the cell viability was assessed using an MTT assay. As shown in Fig. 2, exposure of the cells to ox-LDL for 24 or 48 h significantly increased the cell viability compared with that in the control group. However, icariin inhibited ox-LDL-induced VSMC proliferation in a concentration-dependent manner.

To clarify the effect of icariin on cell-cycle regulation, the cell cycle distribution of ox-LDL-induced VSMCs was assessed using flow cytometry. As shown in Fig. 3, treatment with ox-LDL markedly increased the percentage of VSMCs in S and G2/M phases and correspondingly decreased the percentage of cells in G0/G1 phase. However, pre-treatment with icariin significantly reversed these effects in a concentration-dependent manner. To assess whether the effects of icariin on the cell cycle were associated with the expression of PCNA, western blot analysis was performed. As shown in Fig. 4, the protein expression of PCNA was markedly increased following ox-LDL treatment, which was inhibited by icariin in a dose-dependent manner. The western blot results were in line with the findings of the cell cycle analysis, as ox-LDL enhanced the population of cells in S phase, which was accompanied an increased expression of PCNA, while icariin pre-treatment was able to inhibit these effects in a concentration-dependent manner. These results suggested that icariin inhibits ox-LDL-induced VSMC proliferation by blocking cell cycle progression.

To demonstrate whether icariin inhibited ox-LDL-induced VSMC proliferation by inhibiting the activation of ERK1/2, the phosphorylation levels of ERK1/2 were examined. As shown in Fig. 5, ox-LDL and icariin had no effect on the levels of total ERK1/2. VSMCs incubated with ox-LDL for 24 h showed markedly enhanced ERK1/2 phosphorylation, which was significantly and dose-dependently inhibited by icariin pre-treatment. These results suggested that icariin may reduce ox-LDL-induced proliferation of VSMCs, at least in part, via inhibition of ox-LDL-induced ERK1/2 phosphorylation.