All the cells were cultured in an environment with 5% CO₂ at 37°C. The HepG2 human hepatoma cell line (American Type Culture Collection, Manassas, VA, USA) was cultured in Minimal Essential Medium (MEM; Gibco Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The THP-1 human monocyte cell line (American Type Culture Collection) was cultured in RPMI-1640 medium (Gibco Life Technologies) containing 10% FBS with 10 mmol/l HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich).

Following HepG2 cell growth to a density of 30–50%, the cells were seeded in a 24-well plate with non-serum MEM. Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was applied for the transfection of the plasmids into the cells. The mammalian one-hybridization experiment is a method extensively applied to identify transcription factors at a molecular level. As an agonist of LXR, T0901317 (J&K Scientific Ltd., Shanghai, China) is known to effectively activate the transcriptional activity of the LXRα-ligand binding domain (LBD) (13). Using the mammalian one-hybridization method and T0901317 as a positive control, the effect of various concentrations of magnolol (J&K Scientific Ltd.) on the transcriptional activity of the LXRα-LBD was analyzed in order to identify whether magnolol was able to directly combine with the LXRα-LBD.

In the mammalian one-hybrization experiment, 400 ng pCMX-Gal4-DBD-LXRα-LBD, 400 ng UAS-TK-Luc, and 100 ng reference pRL-SV40 plasmid were transfected (Promega Corporation, Madison, WI, USA). In the transcriptional activation experiment, 400 ng pcDNA3.1a-LXRα, 400 ng pcDNA3.1a-RXRα, 400 ng pGL2-basic-LXRE-Luc plasmids and 100 ng reference plasmid pRL-SV40 were transfected (Promega Corporation). pRL-SV40 was used as the negative control. After cellular transfection for 6 h, the medium was changed to complete medium, 10, 20 or 40 µM magnolol was added and incubated for 18–24 h. The positive control received 50 nM T0901317 and the negative control contained 10 µM DMSO. Following incubation, the cells were washed with phosphate-buffered saline (PBS), and 100 µl lysate was added to each well for cell lysis at 37°C for 20 min. Subsequently, the activity of the firefly luciferase and reference luciferase were detected, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA).

Prior to the PCR experiment, 160 nmol/l phorbol ester (Sigma-Aldrich) was incubated with the THP-1 cells for 24 h to induce cell differentiation into macrophages. The cells were subsequently cultured in non-serum medium containing 50 µg/ml oxidized low-density lipoprotein (Jingmei Biotechnology Co., Nanjing, China) for 48 h, and lipids were phagocyted, then formed foam cells. After 24 h of treatment, the medium in the Petri dish was removed and the cells were washed with PBS. The total RNA was extracted using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instructions. A reverse transcription kit (Takara Biotechnology Co., Ltd.) was used to convert the extracted RNA into cDNA, after which quantitative PCR detection was conducted. The primer sequences were as follows: Human ATP-binding cassette transporter A1 (ABCA1) forward, 5′-GATTGGCTTCAGGATGTCCATGTTGGAA-3′ and reverse, 5′-GTATTTTTGCAAGGCTACCAGTTACATTTGACAA-3′; human ATP binding cassette transporter G1 (ABCG1) forward, GCCACTTTCGTGGGCCCAGTGA-3′ and reverse, 5′-TCTCATCACCAGCTGTGTTGCA-3′. β-Actin was used as the reference gene in the genetic expression experiments, and the β-actin primers were as follows: Forward, 5′-GCGGGAAATCGTGCGTGAC-3′ and reverse, 5′-CGTCATACTCCTGCTTGCTG-3′.

Sample cDNA underwent quantitative PCR amplification using a SYBR fluorescent probe kit (Takara Biotechnology Co., Ltd.). The amplification procedure was as follows: Initial denaturation at 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 20 sec, 72°C for 10 sec and a final extension at 72°C for 10 min.

All experimental data are expressed as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). The statistical significance of differences was analyzed by one way analysis of variance and Dunnett's post hoc test, where P<0.05 was considered to indicate a statistically significant difference.

At 6 h after HepG2 cell transfection with the corresponding plasmid, magnolol was applied at different concentrations, as well as T0901317. After 18–24 h, the detection of luciferase activity was performed. T0901317 was shown to markedly stimulate the transcriptional activity of the LXRα-LBD, while magnolol was shown to dose-dependently increase luciferase activity. These results indicated that magnolol was able to interact with the LXRα-LBD directly (Fig. 1). NRs are ligand-dependent transcription factors, and magnolol was shown to combine with the LXRα-LBD at a molecular scale, demonstrating that magnolol may affect the expression of LXR downstream genes by combining with LXR response elements (LXRE).

To function as transcription factor, LXR is required to form a heterodimer with the RXR, with the combination in the LXRE, in order to initiate downstream genetic expression. A mammalian transcriptional activation experiment was conducted to detect whether magnolol was able to promote the combination of the LXR/RXR heterodimer and LXRE. At 6 h after HepG2 cell transfection with the corresponding plasmids, the cells were treated with magnolol at various concentrations or T0901317 (positive control) for 24 h. As shown in Fig. 2, T0901317 significantly increased the luciferase activity, promoted the combination of the LXR/RXR heterodimer and LXRE, and increased the transcriptional activity of the LXRE. Furthermore, magnolol was shown to dose-dependently increase the transcriptional activity of LXRE. These results indicated that magnolol was able to regulate the combination of the LXR/RXR heterodimer and LXRE, thereby affecting the expression of LXR downstream genes.

ABCA1 and ABCG1 are two essential proteins that mediate cholesterol efflux in macrophages (14). In order to investigate whether magnolol affects the expression of ABCA1 and ABCG1 via the LXR, and subsequently ameliorates AS, the effect of magnolol on the expression of ABCA1 and ABCG1 was investigated in THP-1 macrophage-derived foam cells using quantitative PCR. Magnolol was applied at various concentrations to treat the cells. Magnolol was demonstrated to markedly increase the mRNA expression levels of ABCA1 and ABCG1, and the effect became more significant as the magnolol concentration increased (Fig. 3). Therefore, the results indicated that magnolol dose-dependently regulated the mRNA expression levels of ABCA1 and ABCG1.