Rabbit polyclonal anti-GAPDH immunoglobulin (Ig)G (sc-25778) and rabbit polyclonal anti-B-cell lymphoma (Bcl)-2 IgG (sc-492) antibodies, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2055) secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Icariin (purity≥99.8%) was obtained from the National Institutes for Food and Drug Control (Beijing, China). Oxaliplatin (Eloxatin) was purchased from Sanofi-Aventis (Paris, France). RNase A, RNAiso Plus, the first strand cDNA synthesis kit (cat. no. DRR047) and the SYBR green kit for reverse transctiption-polymerase chain reaction (RT-qPCR) were obtained from Takara Biotechnology Co., Ltd (Dalian, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), high glucose Dulbecco's modified Eagle's medium (DMEM-H), penicillin G, streptomycin, phosphate-buffered saline (PBS), and the 3,3′-diaminoben-zidine (DAB) HRP Color Development kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Washes consisted of three 5 min rinses in PBS, unless otherwise specified.

Human HepG2 cells were cultured in DMEM-H supplemented with 10% FBS, penicillin G (100 U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere with 5% CO₂ and 95% air at 37°C. When the cells reached 80% confluence, after 3 days of culture, they were passaged. The adherent cells were washed and detached using 1 ml 0.25% trypsin-EDTA solution for 2 min. The resuspended cells were then placed into three cell culture flasks for incubation under the same conditions.

The effect of icariin on HepG2 cell proliferation was determined using an MTT assay. Briefly, the HepG2 cells were seeded in 96-well plates at a density of 1×10⁵ cells per well. After 24 h incubation to allow attachment, the medium was replaced with icariin at various concentrations (10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷ or 10⁻⁸ mol/l) for periods of 24, 48 and 72 h. Subsequently, 10 µl MTT (5 mg/ml) was added to each well and the the plates were incubated at 37°C for an additional 4 h. The resulting formazan crystals were solubilized by adding 100 µl DMSO to each well for 20 min. When the crystals had fully dissolved, the plates were read on a micro-plate reader (iMark; Bio-Rad Laboratories Inc., Hercules, CA, USA) at a wavelength of 490 nm. The concentration of drug required to inhibit 50% of cell growth (IC₅₀) was calculated.

Once the optimal conditions of icariin treatment of HepG2 cells had been established (10⁻⁵ M for 72 h), cell cycle analysis was performed on the three flasks of cells cultured with either 10% FBS, oxaliplatin (10 mg/l), or icariin (10⁻⁵ mol/l) for 72 h. The cells were digested using trypsin-EDTA solution, and the resuspended cells were counted (1×10⁶ per flask). The cells were fixed with 70% ethanol at −20°C overnight, washed twice with PBS and incubated with RNaseA (100 mg/ml) in PBS at room temperature for 30 min. The DNA was labeled in the dark using propidium iodide (50 mg/ml) and then washed, following which the cells were analyzed using a FACScalibur flow cytometer (BD Biosciences, San Diego, CA, USA).

The HepG2 cells were cultured in 12-well plates at a density of 1×10⁴ cells/well. FBS (10%), oxaliplatin (10 mg/l), or icariin (10⁻⁵ mol/l⁻¹) were added for 72 h, following which the plates were washed and the cells were fixed with 95% ethanol for 15 min. The cells were then washed again, and incubated in blocking buffer for 15 min at 37°C to prevent non-specific antibody binding. The cells were then incubated in anti-Bcl-2 antibody (1:500) for 1 h at 37°C, and in secondary antibody (1:2,000) for 20 min at room temperature, prior to a final washing step. Staining was visualized using DAB, and nuclei were counterstained with hematoxylin.

The cells were seeded in a 2.5 cm² culture flask. After 24 h, the cells were incubated with FBS (10%), oxaliplatin (10 mg/l) or icariin (10⁻⁵ mol/l⁻¹). After 3 days, the cells were washed with ice-cold PBS and subsequently lysed using a mammalian tissue protein extraction kit (RIPA Lysis Buffer kit; Boster Biological Technology Co. Ltd., Wuhan, China) containing 1 mM phenylmethanesulfonyl fluoride protease inhibitor. The cells were centrifuged at 12,000×g for 10 min at 4°C, and the supernatants were collected. The protein concentration was determined using a bicinchoninic acid kit (Ding Guo, Beijing, China). Equal quantities (50 µg) of protein in the cell extracts were separated on denatured 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (Ding Guo) and the proteins were transferred onto polyvinylidene difluoride membranes (Immobilon; EMD Millipore, Danvers, MA, USA). The membranes were blocked in blocking buffer [5% (w/v) non-fat dry milk (Yi Li, Inner Mongolia, China) in Tris-buffered saline containing 0.1% Tween20 (Boster, Wuhan, China) (TBST)] at 37°C for 60 min and then incubated with anti-Bcl-2 (1:800) or anti-GAPDH antibody (1:1,000) overnight at 4°C. Following four washes with TBST, the membranes were incubated with HRP-conjugated secondary antibody (1:2,000; Protein Tech Group, Inc, Chicago, USA) for 1 h at 37°C. The membranes were then washed five times with TBST. Finally, the immunoblot signals were visualized using enhanced chemilu-minescence reagent (TransGen, Beijing, China). Quantification of proteins was performed using an EC3 Chemi HR 410 imaging system (UVP, Inc., Cambridge, UK).

The cells were incubated in confocal plates at a density of 1×10³ cells per plate. After 3 days, the cells were washed and fixed with 95% ethanol for 15 min, followed by another wash. For imaging analysis of the F-actin filaments, the HepG2 cells were stained with phalloidin-fluorescein isothiocyanate (FITC; 50 mg/l; Invitrogen Life Technologies, Carlsbad, CA, USA) in the dark for 20 min, prior to three washes in PBS. Finally, the samples were analyzed using a confocal laser scanning microscope (Model FV500; Olympus, Tokyo, Japan). Images were captured and quantified using FluoView software (Olympus).

One-way analysis of variance followed by Tukey's post-hoc comparison was used to compare groups. All data are expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated at least three times.

The antiproliferative property of icariin in cultured HepG2 cells was determined using an MTT assay. Icariin significantly inhibited the proliferation of the HepG2 cells at concentrations between 10⁻⁴ and 10⁻⁸ mol/l (Fig. 2). The IC₅₀ was observed at ~10⁻⁵ mol/l at 72 h, therefore, this concentration was selected for use in the subsequent experiments.

The oxaliplatin group and the icariin group exhibited significantly higher proportions of the cell population in the G₀/G₁ phase, compared with the control group (P<0.05; Fig. 3). The proportion of HepG2 cells in the oxaliplatin (24.19%) and icariin (21.07%) groups at the S phase were significantly lower, compared with that in the control group (28.62%; P<0.05 and P<0.01, respectively). No significant difference in cell cycle distribution was observed between the oxaliplatin and icariin groups.

Bcl-2, an anti-apoptotic molecule, can be used as a marker for cell apoptosis. Therefore, the presents study examined expression of Bcl-2 in HepG2 cells using immunocytochemical and western blot analyses. The expression levels of Bcl-2 were significantly lower in the oxaliplatin and icariin groups, compared with the control group (P<0.01; Fig. 4). Furthermore, the expression of Bcl-2 in the icariin-treated cells was lower than that in the oxaliplatin-treated cells (P<0.01). Western blot analysis confirmed the results of the immunohistochemical analysis, with a lower expression level of Bcl-2 observed in the oxaliplatin and icariin groups, compared with the control (P<0.05 and P<0.01, respectively; Fig. 5). Icariin treatment led to a sharp decrease in the protein expression of Bcl-2, compared with oxaliplatin treatment (P<0.01).

Confocal microscopy was used to determine the effects of icariin on F-actin in the HepG2 cells (Fig. 6). In the untreated HepG2 cell control group, long-form and regular F-actin filaments were observed, however, polymerization of F-actin filaments was noticeably disrupted following 3 days of exposure to either oxaliplatin or icariin.