Matrine (purity, >99%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco's modified Eagle's medium, fetal bovine serum (FBS) and Lipofectamine Plus were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The pGL3-NF-κB vector and the luciferase assay system were provided by Promega (Madison, WI, USA), and the pCMV-β-gal vector was obtained from Lonza (Walkersville, MD, USA). Antibodies against ICAM-1 (mouse monoclonal) and VCAM-1 (mouse monoclonal) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA) and antibodies against the inhibitor of NF-κB (IκB-α; rabbit polyclonal), p65 (rabbit polyclonal), c-Jun N-terminal kinase (JNK; rabbit polyclonal), phospho-JNK (p-JNK; rabbit polyclonal), extra-cellular signal-regulated kinase (ERK; rabbit monoclonal), p-ERK (rabbit polyclonal), p38 (rabbit polyclonal), p-p38 (rabbit polyclonal), Akt, p-Akt, lamin A (rabbit polyclonal) and β-actin (rabbit polyclonal) were purchased from Abcam Inc. (Cambridge, MA, USA). PCR primers and PCR premix were purchased from Bioneer Corporation (Daejeon, South Korea). phosphate-buffered saline (PBS), tris-buffered saline (TBS) and Tween 20 were purchased from Sigma-Aldrich. (St. Louis, MO, USA).

HASMCs were purchased from Clonetics Corp. (San Diego, CA, USA) and cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, and 5% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) with 2 ng/ml basic fibroblast growth factor, 10 ng/ml recombinant human epidermal growth factor and 5 µg/ml insulin (all from Sigma-Aldrich).

The effects of matrine on the proliferation of HASMCs were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HASMCs were seeded onto 96-well plates (1×10⁴ cells/well) and subsequently treated with various concentrations of matrine (0, 10, 50 and 100 µg/ml). Following 72 h of incubation, MTT (0.5 mg/ml; Sigma-Aldrich) was added to each well. After incubation for 4 h, the supernatant was removed and 0.1% dimethylsulfoxide (Sigma-Aldrich) was added to dissolve the formazan crystals. The absorbance at 550 nm was measured using a microplate reader 3350 (Bio-Rad Laboratories, Hercules, CA, USA) and the percentage of viable cells compared with that in the control group was calculated.

RNA extraction was performed with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequently, RNA was reverse-transcribed in a 20-ml reaction system using an Advantage RT kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) according to the supplier's recommended protocol. mRNA levels were quantified by RT-qPCR using SYBRGreen Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a 30 ng template in a 20 µl reaction mixture. For PCR amplification, the following primers were used: VCAM-1 forward, 5′-CAA AGG TGG ATC AGA TTC AAG-3′ and reverse, 5;-GGT GAG CAT TAT CAC CCA GAA-3′; ICAM-1 forward, 5′-CAA AGG TGG ATC AGA TTC AAG-3′ and reverse, 5;-GGT GAG CAT TAT CAC CCA GAA-3′; GAPDH forward, 5′-CAA AGG TGG ATC AGA TTC AAG-3′ and reverse, 5;-GGT GAG CAT TAT CAC CCA GAA-3′. The PCR cycling program was 95°C for 3 min, followed by 28 cycles of 95°C for 20 sec, 60°C for 20 sec and 72°C for 15 sec, and a final extension at 72°C for 5 min. The levels of individual gene mRNA transcripts were initially normalized to the control β-actin. Subsequently, the differential expression of these genes was analyzed using the 2^−ΔΔCq method (14).

HVSMCs were cultured and treated with matrine as described above and subsequently subjected to western blot analysis. After treatment, the cells were washed twice with PBS and suspended in 70 µl of Buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF and Protease Inhibitor Cocktail (Sigma-Aldrich)] and incubated on ice. After 15 min, 0.5% Nonidet P (NP)-40 was added to lyse the cells, which were vortexed for 1 sec. Then, cytosolic cell extracts were obtained after centrifuging at 1500 × g for 10 min at 4°C. The collected nuclei were resuspended in 50 µl of Buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 25% v/v glycerol, 0.5 mM PMSF and Protease Inhibitor Cocktail] and incubated on ice for 20 min with intermittent agitation. Nuclear cell extracts were recovered after centrifugation for 10 min at 13,000 × g at 4°C. The protein concentration in the cell extracts was then determined using the Bradford protein dye reagent (Bio-Rad Laboratories, Inc.). Proteins (30 µg/lane) were separated on 10% sodium dodecyl sulfate polyacrylamide gels (Bio-Rad Laboratories) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in TBST buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1% Tween 20) and incubated with the appropriate primary antibodies overnight at 4°C. Subsequent to washing the membranes three times with phosphate-buffered saline, 5 min per wash, containing 0.1% (v/v) Tween 20, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (MyBioSource, Inc., San Diego, CA, USA) for 1 h, followed by visualization of the antibodies using enhanced chemiluminescence detection reagents. The blots were developed using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK). Semi-quantitative determination of protein levels was performed using Image-Pro Plus software (NIH Image J 1.61; Media Cybernetics, Rockville, MD, USA).

The cells (1×10⁵ cells/ml) were plated into each well of a 6-well plate. The cells were transiently co-transfected with the plasmids, pGL3-NF-κB and pCMV-β-gal using Lipofectamine Plus according to the manufacturer's protocol. Briefly, a transfection mixture containing 0.5 µg pGL3-NF-κB and 0.2 µg pCMV-β-gal was mixed with the Lipofectamine Plus reagent and added to the cells. After 4 h, the cells were pretreated with matrine, and then lysed with 200 µl of lysis buffer (24 mM Tris-HCl (pH 7.8), 2 mM dithiotreitol, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) and 10 µl of cell lysates were used for luciferase activity assay. The luciferase and β-galactosidase activities were determined. The values shown represent an average of three independent transfections, which were normalized with β-galactosidase activity. Each transfection was performed in triplicate and experiments were repeated three times.

ROS levels were determined according to the method of a previous study (15). 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CMH₂DCFDA; Molecular Probes, Eugene, OR, USA) was used to determined intracellular ROS levels using flow cytometry. Following pre-treatment of HASMCs (3×10⁶ cells/ml) with various concentrations of matrine for 2 h, cells were incubated with TNF-α (10 ng/ml) for 4 h. The cells were then stained with 5 µM CMH₂-DCFDA for 15 min at 37°C. The cells were kept in the dark on ice and at least 10,000 cells for each sample were analyzed using a Becton Dickinson FACSCalibur (BD Biosciences, San Jose, CA, USA). Changes in the levels of intracellular ROS are expressed as a percentage of TNF-α-stimulated, matrine-untreated cells.

Values are expressed as the mean ± standard error of the mean. Data from different groups were compared using a Student's t-test or one-way analysis of variance followed by Dunnett's test. Statistical analysis was performed using SPSS (version 12.5S; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To examine the effect of matrine on cell viability, HASMCs were treated with various concentrations of matrine for 8 h and subjected to an MTT assay. As shown in Fig. 1, no significant cytotoxicity of matrine was observed. These observations indicated that matrine had no effect on the viability of HASMCs.

To examine whether matrine affects TNF-α-mediated induction of adhesion molecules, HASMCs were pre-treated with various concentrations of matrine for 2 h, followed by stimulation with TNF-α (10 ng/ml) and subjected to RT-qPCR analysis. As shown in Fig. 2A and B, treatment with TNF-α induced the mRNA expression of VCAM-1 and ICAM-1 on HASMCs. However, matrine significantly inhibited TNF-α-induced mRNA expression of VCAM-1 and ICAM-1 in a concentration-dependent manner. Consistent with these results, western blot analysis showed that matrine obviously suppressed TNF-α-induced protein expression of VCAM-1 and ICAM-1 in a concentration-dependent manner (Fig. 2C). These results suggested that matrine effectively blocks TNF-α-induced expression of VCAM-1 and ICAM-1.

Activation of NF-κB is linked with the development of vascular damage; furthermore, transcription factors are known to mediate the expression of adhesion molecules (16). Therefore the present study examined the effects of matrine on NF-κB-mediated transcriptional activation. HASMCs were treated with various concentrations of matrine for 2 h and subsequently stimulated with TNF-α for 4 h. Transcriptional activation assays were then employed to determine whether matrine affects NF-κB-dependent transcription. Stimulation with TNF-α obviously increased luciferase activity, while matrine significantly prevented this effect (Fig. 3A). Furthermore, the expression of NF-κB p65 protein was detected by western blot analysis to clarify the inhibitory action of matrine. As shown in Fig. 3B, pre-treatment of HASMCs with matrine significantly decreased the nuclear levels of NF-κB p65, while simultaneously increasing its cyto-solic levels. Furthermore, the effects of matrine on IκB protein in TNF-α-stimulated HASMCs were determined. TNF-α caused a significant degradation of IκBα at 30 min, which was inhibited by matrine (Fig. 3C). These results suggested that matrine impedes TNF-α-induced NF-κB activation.

MAPK signaling is involved in the regulation of adhesion-molecule expression (16). Therefore, the present study investigated the effects of matrine on TNF-α-induced phosphorylation of MAPKs in HAMSCs. As shown in Fig. 4, TNF-α significantly increased the activation of p38/MAPK, ERK1/2 and JNK in matrine-untreated cells. However, matrine concentration-dependently inhibited TNF-α-induced phosphorylation of MAPKs in HAMSCs.

As it has been reported that TNF-α-induced ROS production activates NF-κB in vascular cells, the present study investigated the effect of matrine on the production of TNF-α-induced ROS production in HAMSCs (17). HAMSCs were pre-treated with matrine for 2 h and then stimulated with TNF-α. As shown in Fig. 5, matrine significantly reduced the production of TNF-α-induced ROS in a concentration-dependent manner. The production of ROS was reduced to ~50% by the highest concentration of matrine (100 µg/ml).