Human aortic vascular smooth muscle cell line (HA-VSMCs; cat. no. CRL-1999™) was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified eagle medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 10 ng/ml recombinant human epidermal growth factor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 2 ng/ml basic fibroblast growth factor (Sigma-Aldrich; Merck KGaA), and 5 µg/ml insulin (Sigma-Aldrich; Merck KGaA) in a humidified atmosphere containing 5% CO₂ for 7 d at 37°C. HASMCs were subsequently incubated with TNF-α (10 ng/ml) for different times (30 min or 4 h), with or without pretreatment with, 5 or 10 µM cordycepin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 2 h at 37°C.

Cell proliferation was determined via MTT assay. In brief, HA-VSMCs were seeded in 96-well plates at a density of 1×104 cells/well and cultured in DMEM at 37°C until they reached 80% confluence. Then, MTT (0.2 mg/ml) was added to each well and incubated for 4 h at 37°C. The supernatant was removed and the formazan crystals were dissolved in dimethylsulfoxide. The absorbance was measured using a spectrophotometer at 550 nm (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). HA-VSMCs treated with DMEM were used as a control.

For DNase treatment 2 units of DNase I polymerase (Invitrogen; Thermo Fisher Scientific, Inc.) were used per µg of total RNA at 37°C for 30 min. Total RNA was extracted from HA-VSMCs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Approximately 5 µg total RNA for each sample were reverse-transcribed using an oligo-(dT) primer and M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. qPCR was performed in a final volume of 10 µl, which contained 5 µl SsoFast™ EvaGreen Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1 µl cDNA (1:50 dilution) and 2 µl the forward and reverse primers (1 mM). qPCR was performed with the ABI Prism 7500 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR-Green real-time PCR Master Mix kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. Primers were designed as follows; VCAM-1 forward, 5′-CAAAGGTGGATCAGATTCAAG-3′ and reverse, 5′-GGTGAGCATTATCACCCAGAA-3′; ICAM-1 forward, 5′-CAAAGGTGGATCAGATTCAAG-3′ and reverse, 5′-GGTGAGCATTATCACCCAGAA-3′; and β-actin forward, 5′-AGAAAATCTGGCACCACACC-3′ and reverse, 5′-TAGCACAGCCTGGATAGCAA-3′. The PCR cycling program was 95°C for 5 min, then 35 cycles of 95°C for 20 sec, 58°C for 20 sec and 72°C for 20 sec, and a final extension at 72°C for 5 min. β-actin was used as the internal reference gene. The relative expression levels were calculated using the 2^−ΔΔCq method, and the target gene was normalized to β-actin (13).

HA-VSMCs were homogenized and lysed with radioimmunoprecipitation assay lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.; 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM sodium vanadate and protease inhibitor). Lysates were sonicated for 5 sec on ice and centrifuged at 6,000 × g for 5 min at 4°C. Supernatants were collected and the protein concentration was detected using a Bio-Rad protein assay kit (cat. no. 500-0002; Bio-Rad Laboratories, Inc.). A total of 30 µg protein was separated by 10% SDS-PAGE followed by electro-blotting-mediated transference onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking in Tris-buffered saline buffer (50 mmol/l NaCl, 10 mmol/l Tris, pH 7.4) containing 5% nonfat milk for 2 h at room temperature, the membranes were subsequently incubated overnight at 4°C with anti-VCAM-1 (1:2,000; cat. no. ab7224, Abcam, Cambridge, UK), anti-ICAM-1 (1:1,000; cat. no. sc-8439, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phosphorylated (p)-p38 (1:1,500; cat. no. sc-166182, Santa Cruz Biotechnology, Inc.), anti-p38 (1:1,500; cat. no. sc-81621, Santa Cruz Biotechnology, Inc.), anti-p-extracellular signal-regulated kinase (ERK)1/2 (1:2,000; cat. no. sc-136521, Santa Cruz Biotechnology, Inc.), anti-ERK1/2 (1:2,000; cat. no. sc-514302, Santa Cruz Biotechnology, Inc.), anti-p- c-Jun N-terminal kinase (JNK; 1:3,000; sc-293136, Santa Cruz Biotechnology, Inc.), anti-JNK (1:3,000; cat. no. sc-137020, Santa Cruz Biotechnology, Inc.), anti-p-Akt (1:1,000; sc-52940, Santa Cruz Biotechnology, Inc.), anti-Akt (1:1,000; cat. no. sc-5298, Santa Cruz Biotechnology, Inc.), anti-NF-κB p65 (1:2,000; ca. no. sc-8008, Santa Cruz Biotechnology, Inc.) anti-IκBα (1:1,000; cat. no. sc-1643, Santa Cruz Biotechnology, Inc.) and anti-β-actin (1:1,500; cat. no. sc-8432, Santa Cruz Biotechnology, Inc.) primary antibodies. Membranes were subsequently washed three times with PBS containing 0.1% (v/v) Tween-20 (Invitrogen) for 10 min and incubated with horseradish peroxidase conjugated goat anti-mouse IgG-horse radish peroxidase secondary antibodies (1:3,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature, followed by exposure using enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, Inc.). BandScan 5.0 software (Glyko, Inc., Novato, CA, USA) was used for the quantification of proteins following western blot analysis. All experiments were repeated at least three times.

Data was analyzed using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean. Statistical evaluation was performed using Student's t-test or one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of cordycepin on cell viability, an MTT assay was performed. The results revealed no significant difference between the experimental and control groups following of HA-VSMCs with 1–10 µM cordycepin (Fig. 1).

The effect of cordycepin on the expression of adhesion molecules in HA-VSMCs was investigated using RT-qPCR and western blot analysis. Treatment with TNF-α significantly induced the expression of VCAM-1 and ICAM-1 mRNA (Fig. 2A and B). Cordycepin, however, significantly inhibited the TNF-α-induced expression of VCAM-1 and ICAM-1 mRNA in a concentration-dependent manner (Fig. 2A and B). Similarly, western blot analysis indicated that cordycepin significantly suppressed the TNF-α-induced protein expression of VCAM-1 and ICAM-1 in a concentration-dependent manner (Fig. 2C).

MAPK and Akt are two important intracellular pathways involved in cell adhesion (14), and so in the present study various members of the MAPK and Akt signal networks were detected via western blotting (Fig. 3A). It was demonstrated that the phosphorylation levels of p38 MAPK (Fig. 3B), ERK1/2 (Fig. 3C), JNK (Fig. 3D), and Akt (Fig. 3E) were significantly increased in TNF-α-stimulated HA-VMSCs, compared with the control. However, cordycepin treatment significantly inhibited the TNF-α-induced phosphorylation of MAPKs and Akt in HA-VMSCs (Fig. 3B-E).

Activation of NF-κB serves an important role in the development of vascular damage, and transcription factors have been known to be important mediators of adhesion molecule expression (15). Therefore, in the present study the effect of cordycepin on NF-κB transcriptional activation was investigated. Pre-incubation of HA-VMSCs with cordycepin induced a dose-dependent decrease in NF-κB p65 translocation to the nuclear fraction (Fig. 4A). The effect of cordycepin on IκB protein in TNF-α-stimulated HA-VSMCs was subsequently investigated, suggesting that that the cordycepin only treatment affects the degradation of IκBα. The results demonstrated that cordycepin markedly reduced the TNF-α-induced degradation of IκBα (Fig. 4B).