The human ASMC line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium containing 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in a humidified atmosphere of 5% CO₂ at 37°C. ASMCs in passages 9–20 were used for the experiments presented in this study.

Cell viability was measured using an MTT assay. In brief, ASMCs at a density of 1×10⁴ cells/well were seeded in a 96-well plate and grown in DMEM medium containing 10% FBS for 24 h. ASMCs were pretreated with PF (10, 20 and 40 nM; Sigma-Aldrich; Merck KGaA) for 1 h prior to stimulation with PDGF-BB (10 ng/ml; Sigma-Aldrich; Merck KGaA) for 24, 48 or 72 h. Then, MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to each well and incubated for 4 h at 37°C in 5% CO₂. Subsequently, the supernatants were removed, and 150 µl of dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance at 450 nm was measured using a Spectra Max 190 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

Cell migration was examined using transwell chambers. In brief, following treatment as described above, ASMCs at a density of 5×10⁴ cells/well suspended in 0.1% FBS medium were added into the upper chamber. DMEM medium (500 µl) containing 10% FBS, with or without PDGF-BB, was added into the lower chamber. Following incubation at 37°C for 24 h, cells migrated to the lower surface of the transwell membrane were stained with hematoxylin and eosin (Sigma-Aldrich; Merck KGaA), and the mean number of cells per four fields of view was counted under a light microscope. Migration was measured as the number of cells/field.

Total RNA was extracted from ASMCs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. cDNA was synthesized from the extracted RNA (3 µg) using the SuperScript First-Strand cDNA Synthesis SuperMix kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR analysis was performed using a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with Fast Start Universal SYBR Green Master mix (Roche Diagnostics, Basel, Switzerland). The specific primers were: α-smooth muscle actin (α-SMA) sense, 5′-CTATTCCTTCGTGACTACT-3′ and antisense, 5′-ATGCTGTTATAGGTGGTGGTT-3′; β-actin sense, 5′-TTAGTTGCGTTACACCCTTTC-3′ and antisense, 5′-ACCTTCACCGTTCCAGTTT-3′. The PCR cycling program was 95°C for 3 min, then 35 cycles of 94°C for 20 sec, 59°C for 30 sec and 72°C for 20 sec, and a final extension at 72°C for 5 min. β-actin was used as a normalization control and the results were analyzed by the method of 2^−ΔΔcq (15).

ASMCs were washed with PBS and lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nantong, China). The cell lysate supernatants were harvested by centrifugation at 10,000 × g for 10 min at 4°C. The protein concentration was determined using a Bradford protein assay. Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Sigma-Aldrich; Merck KGaA). Following blocking with 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies targeting α-SMA (1:2,500; cat. no. sc-53142), phosphoinositide 3-kinase (PI3K, 1:2,000; cat. no. sc-365290), phosphorylated (p)-PI3K (1:2,500; sc-293115), AKT serine/threonine kinase 1 (Akt; (1:1,000; cat. no. sc-5298), p-Akt (1:1,000; cat. no. sc-52940) and GAPDH (1:3,000, cat. no. sc-59540; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Then, the membranes were washed with TBS containing 0.1% Tween for 10 min and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,500, cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Protein bands were evaluated by enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). The optical densities of the bands were quantified using Gel-Pro Analyzer v4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

The results were analyzed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). All experiments were performed at least in triplicate and results are expressed as mean ± standard deviation. Statistical significance was analyzed with the one-way analysis of variance or the Student's two-tailed t-test followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

First, the effect of PF treatment on ASMC growth was examined using the MTT assay. As indicated in Fig. 1, PDGF-BB stimulation significantly induced growth in ASMCs over the course of 72 h, compared with the control group. However, the PDGF-BB-induced effect on ASMC growth was inhibited by PF pretreatment in a concentration-dependent manner (Fig. 1).

ASMC migration is hypothesized to be a key event in the pathogenesis of asthma (16). Thus, the effect of PF treatment on ASMC migration in response to PDGF-BB stimulation was examined. The results of transwell migration assays demonstrated that stimulation with PDGF-BB greatly promoted the migration of ASMCs, compared with the control group (Fig. 2). However, PF pretreatment partially reversed this effect in a dose-dependent manner (Fig. 2).

α-SMA is a major constituent of the contractile apparatus of ASMCs, and its expression has been associated to the progression of asthma (17). Therefore, the effect of PF treatment on α-SMA expression in ASMCs was investigated. As demonstrated in Fig. 3A, PDGF-BB stimulation in ASMCs significantly increased the mRNA expression levels of α-SMA, compared with the control group. However, PF pretreatment partially inhibited the PDGF-BB-induced α-SMA mRNA overexpression (Fig. 3A). Similarly, the results of western blot analysis demonstrated that PF pretreatment suppressed PDGF-BB-induced α-SMA protein overexpression in ASMCs, compared with cells treated with PDGF-BB alone (Fig. 3B).

To explore the signaling mechanisms involved in the effects of PF on ASMC growth and migration, the phosphorylation status of PI3K and Akt was investigated by western blotting. As demonstrated in Fig. 4, PDGF-BB stimulation significantly activated PI3K and Akt phosphorylation in ASMCs compared with the control group. This activation was partially reversed by PF pretreatment, with phosphorylation levels for both PI3K and Akt significantly reduced compared with cells treated with PDGF-BB alone (Fig. 4).