HUVECs were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and were seeded into 96-well plates at a density of 1×10⁴ cells/well and incubated for 12 h (37°C, 5% CO₂) in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Hangzhou Sijiqing Bioengineering Material Co., Ltd., Hangzhou, China), 1% penicillin and 1% streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Following pretreatment with 0, 10, 20 and 40 µM icariin (Sichuan Weike Biotechnology Co., Ltd., Chengdu, China) for 24 h at 37°C, cells were treated for 24 h with 100 µg/ml ox-LDL (Guangzhou Yiyuan Biological Technology Co., Ltd., Guangzhou, China).

Based on a previous report (17), the cells were maintained for 24 h in serum-free DMEM to achieve cell cycle synchronization prior to their treatments with icariin and ox-LDL. Following pretreatment with icariin and ox-LDL, MTT reagent (0.5 mg/ml) was added to the cells at a density of 1×10⁴ cells/well and they were incubated for 4 h at 37°C. Subsequently, the precipitate was dissolved in 150 µl dimethyl sulfoxide, and the optical density of the supernatant was measured at a wavelength of 490 nm.

In the present study, the apoptotic ability was evaluated using an Annexin V fluorescein isothiocyanate (FITC) kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing China), FACSCalibur (BD Biosciences, San Jose, CA, USA) and ModFit LT V3.3.11 software (Verity Software House Inc., Topsham, ME, USA). Cells were washed with PBS and centrifuged for 5 min at 800 × g at 4°C, and the treated cells were resuspended in 200 ml 1X Annexin binding buffer and harvested. Subsequently, cells were stained with 5 ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) and 2.5 ml Annexin V/FITC, and protected from light for 15 min at 37°C.

HUVECs were seeded into 6-well plates at a density of 1×10⁴ cells/well, treated as aforementioned, harvested and lysed for 30 min in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). Following centrifugation for 20 min at 13,000 × g and 4°C, the supernatants were analyzed using a bicinchoninic acid assay. Equal amounts of protein samples (50 µg) were loaded onto a 15% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes, which were blocked with 5% fat-free milk for 1 h at room temperature. At 4°C membranes were incubated with rabbit anti-human monoclonal antibodies against caspase-3 (1:1,000; cat. no. ab23021; Abcam, Cambridge, UK), anti-Bcl-2 (1:1,000; cat. no. ab47482; Abcam) or anti-GAPDH (1:1,000; Sigma-Aldrich; Merck KGaA; cat. no. sab4300645) overnight, followed by horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. sc45101; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at ambient temperature. The bands were visualized by enhanced chemiluminescence/X-ray films (GE Healthcare, Little Chalfont, UK) and were analyzed using ImageJ version 1.46 (National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted from HUVECs with TRIzol^™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 5 µg total RNA was reverse transcribed using a PrimeScript RT Master Mix kit (Takara Biotechnology Co., Ltd., Dalian, China), and RT-qPCR was performed using SYBR^® Premix Ex Taq (Beijing Transgen Biotech Co., Ltd., Beijing, China) and the following program: Denaturing at 95°C for 10 sec, annealing at 60°C for 15 sec and extension at 72°C for 30 sec (40 cycles). GAPDH was used as a housekeeping gene. The following oligonucleotide primers were used: Caspase-3 forward, 5′-GTGGAATTGATGCGTGATG-3′ and reverse, 5′-GGAATCTGTTTCTTTGCATG-3′; Bcl-2 forward, 5′-GGTGCCACCTGTGGTCCACCT-3′ and reverse, 5′-CTTCACTTGTGGCCCAGATAGG-3′; and GAPDH forward, 5′-GTTACCAGGGCTGCCTTCTC-3′ and reverse, 5′-GATGGTGATGGGTTTCCCGT-3′. Relative quantification was calculated using the 2^−ΔΔCq method (22) and the results were normalized to those of GAPDH.

The SPSS 19.0 program (IBM, Corp., Armonk, NY, USA) was used. Each independent experiment was performed in triplicate. One-way analysis of variance followed by a Tukey's post-hoc analysis was used, and the data are presented as the mean ± standard error. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether icariin exerts a protective effect against injury, the present study analyzed the viability of HUVECs treated with ox-LDL via an MTT assay. Treatment with ox-LDL significantly decreased the viability of HUVECs compared with control cells (Fig. 2), while icariin mitigated this decrease. These findings suggested that icariin may have exerted protective effects against injury within HUVECs stimulated with ox-LDL.

To determine the effect of icariin on cellular apoptosis, Annexin-V and PI double staining was performed. The present study investigated the apoptosis rate of HUVECs treated with ox-LDL using flow cytometry. The apoptosis rate significantly increased when HUVECs were treated with ox-LDL compared with the control group (Fig. 3). Additionally, pretreatment with icariin significantly mitigated this effect, the improvement was more significant at 40 µM icariin compared with the other assayed concentrations. These findings indicated that icariin markedly inhibited apoptosis in HUVECs stimulated with ox-LDL.

To investigate the mechanism underlying the protective effects of icariin on ox-LDL-induced cellular apoptosis, the relative gene and protein expression levels of Bcl-2 and caspase-3 were quantified via RT-qPCR and western blot analysis, respectively. Treatment of HUVECs with ox-LDL significantly increased caspase-3 and decreased Bcl-2 expression at the protein (Fig. 4) and mRNA level (Fig. 5). Conversely, icariin pretreatment significantly suppressed these alterations. The results indicated that icariin exerts antiapoptotic effects by downregulating caspase-3 mRNA and protein expression levels while upregulating those of Bcl-2.